Wall Glycoproteins Research Articles

The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of theChlamydomonascell wall

Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.

Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase. Apart from the catalytic Stt3p, the roles of the subunits are ill defined. Here we describe functional investigations of the Ost3/6p components of the yeast enzyme. We developed novel analytical tools to quantify glycosylation site occupancy by enriching glycoproteins bound to the yeast polysaccharide cell wall, tagging glycosylated asparagines using endoglycosidase H glycan release, and detecting peptides and glycopeptides with LC-ESI-MS/MS. We found that the paralogues Ost3p and Ost6p were required for efficient glycosylation of distinct defined glycosylation sites. Our results describe a novel method for relative quantification of glycosylation occupancy in the genetically tractable yeast system and show that eukaryotic oligosaccharyltransferase isoforms have different activities toward protein substrates at the level of individual glycosylation sites.

Genomic and expression analysis of glycosyl hydrolase family 35 genes from rice (Oryza sativa L.)

BackgroundMany plant β-galactosidases (Bgals) have been well characterized and their deduced biological functions mainly involve degradation of structural pectins, xyloglucans or arabinogalactoproteins in plant cell walls. However, gene multiplicity in glycosyl hydrolase family 35 (GH35), to which these proteins belong, implies diverse functions. In this study, the gene multiplicity, apparent evolutionary relationships and transcript expression of rice Bgal genes were examined, in order to predict their biological functions.ResultsFifteen rice Bgal genes were identified in the plant genome, one of which encodes a protein similar to animal Bgals (OsBgal9), and the remaining 14 fall in a nearly plant-specific subfamily of Bgals. The presence of both classes of Bgals in bryophytes, as well as vascular plants, suggests both gene lineages were present early in plant evolution. All 15 proteins were predicted to contain secretory signal sequences, suggesting they have secretory pathway or external roles. RT-PCR and database analysis found two distinct lineages to be expressed nearly exclusively in reproductive tissues and to be closely related to Arabidopsis Bgals expressed most highly in flower and pollen. On the other hand, OsBgal6 is expressed primarily in young vegetative tissues, and alternative splicing in panicle prevents its production of full-length protein in this reproductive tissue. OsBgal11 also showed alternative splicing to produce different length proteins. OsBgal13 produced by recombinant expression in Escherichia coli hydrolyzed α-L-arabinoside in addition to β-D-galactoside and β-(1→3)-, β-(1→4)- and β-(1→6)- linked galacto-oligosaccharides.ConclusionRice GH35 contains fifteen genes with a diversity of protein sequences, predicted locations and expression and splicing patterns that suggest that OsBgals enzymes may play a variety of roles in metabolism of cell wall polysaccharides, glycoproteins and glycolipids.

Role of the serine-rich surface glycoprotein GspB of Streptococcus gordonii in the pathogenesis of infective endocarditis

The direct binding of bacteria to platelets is a central interaction in the pathogenesis of infective endocarditis. GspB is a serine-rich, cell wall glycoprotein of Streptococcus gordonii that mediates the binding of this organism to human platelets in vitro. To assess the contribution of this adhesin to the pathogenesis of endocarditis, we compared the virulence of S. gordonii M99 (which expresses GspB) with an isogenic, gspB mutant (PS846) in two rat models of endovascular infection. In the first group of experiments, animals were infected intravenously with M99 or PS846, and sacrificed 72 h later, to assess levels of bacteria within cardiac vegetations, kidneys, and spleens. When inoculated with 10 5 CFU, rats infected with PS846 had significantly lower densities of organisms within vegetations (mean: 3.84 log 10 CFU/g) as compared with M99-infected rats (6.67 log 10 CFU/g; P < 0.001). Marked differences were also seen in rats co-infected with M99 and PS846, at a 1:1 ratio. While M99 was found at high levels within vegetations, kidneys and spleens (mean log 10 CFU/g: 6.62, 5.07 and 4.18, respectively) PS846 was not detected within these tissues. Thus, platelet binding by GspB appears to be a major interaction in the pathogenesis of endocarditis due to S. gordonii.

Phenotype analysis of Saccharomyces cerevisiae mutants with deletions in Pir cell wall glycoproteins

Proteins with internal repeats (Pir) belong to a minor group of covalently linked yeast cell wall proteins. They are not essential for viability but important for cell wall strength, reduced permeability against plant antifungal enzymes and maintenance of osmotic stability. Here we show the importance of Pir proteins of Saccharomyces cerevisiae for growth at low pH and in presence of various inhibitors. Cell wall analysis of Deltapir1,2,3,4 deletion strain revealed slightly increased chitin content and changes in relative proportion of alkali-soluble and insoluble glucan and chitin fractions. Activation of the cell wall integrity pathway was indicated by increased levels of double phosphorylated Mpk1p/Slt2p in the pir deletants.

The Expression of a Bean PGIP in Transgenic Wheat Confers Increased Resistance to the Fungal Pathogen Bipolaris sorokiniana

A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plant's ability to inhibit the pathogen's capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheat's defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens.

Posttranscriptional regulation ofFLO11upon amino acid starvation inSaccharomyces cerevisiae

Various starvation conditions cause adhesive growth of haploid cells or pseudohyphae formation of diploid cells of Saccharomyces cerevisiae. For the genetic Sigma1278b background, these morphological changes depend on the expression of the gene encoding the cell wall glycoprotein Flo11p, which is increased during nutritional limitations. Deletion of the genes encoding the transcriptional coactivators Rsc1p or Gcn5p impairs FLO11 transcription, which consequently leads to a loss of both haploid invasive growth and diploid pseudohyphae development upon glucose and nitrogen limitation, respectively. In contrast, amino acid starvation induces FLO11-dependent adhesive growth of the rsc1Delta and gcn5Delta strains although FLO11 transcription remains very low. The double deletion strain rsc1Deltaflo11Delta, however, does not grow adhesively, suggesting that the adhesion of the rsc1Delta strain at amino acid starvation is still FLO11-dependent. The FLO11prom-lacZ-encoded beta-galactosidase activities of the rsc1Delta and gcn5Delta mutant strains increase manifold upon amino acid starvation. It is therefore concluded that low levels of FLO11 transcripts are essential and sufficient for derepression of FLO11 expression and adhesive growth during amino acid starvation. A posttranscriptional control is assumed to be behind this phenomenon that permits the increased FLO11 expression from low FLO11 transcript abundances.

Conserved processes and lineage-specific proteins in fungal cell wall evolution.

The cell wall is a defining organelle that differentiates fungi from its sister clades in the opisthokont superkingdom. With a sensitive technique to align low-complexity protein sequences, we have identified 187 cell wall-related proteins in Saccharomyces cerevisiae and determined the presence or absence of homologs in 17 other fungal genomes. There were both conserved and lineage-specific cell wall proteins, and the degree of conservation was strongly correlated with protein function. Some functional classes were poorly conserved and lineage specific: adhesins, structural wall glycoprotein components, and unannotated open reading frames. These proteins are primarily those that are constituents of the walls themselves. On the other hand, glycosyl hydrolases and transferases, proteases, lipases, proteins in the glycosyl phosphatidyl-inositol-protein synthesis pathway, and chaperones were strongly conserved. Many of these proteins are also conserved in other eukaryotes and are associated with wall synthesis in plants. This gene conservation, along with known similarities in wall architecture, implies that the basic architecture of fungal walls is ancestral to the divergence of the ascomycetes and basidiomycetes. The contrasting lineage specificity of wall resident proteins implies diversification. Therefore, fungal cell walls consist of rapidly diversifying proteins that are assembled by the products of an ancestral and conserved set of genes.

The Chaotrope-soluble Glycoprotein GP2 Is a Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.

Rapid adhesion of Stagonospora nodorum spores to a hydrophobic surface requires pre-formed cell surface glycoproteins

Adhesion of fungal pathogens to leaf surfaces is an important first step in the infection process. Previous work on Stagonospora nodorum, a major necrotrophic pathogen of wheat and other cereals, has shown that conidia attach rapidly to a hydrophobic surface and this is followed by the active secretion of extracellular matrix material to consolidate adhesion. In this paper the role of pre-formed spore surface glycoproteins in the rapid adhesion of S. nodorum conidia to an artificial surface, polystyrene, has been investigated. Sodium dodecyl sulphate (SDS) and the enzymes chitinase and lyticase have been used to release cell wall glycoproteins from spores and these have been identified using SDS polyacrylamide gel electrophoresis (PAGE) and Western blotting. Labelling with fluorescently tagged lectins has also been used to study the spore surface. The results show that there are a small number of glycoproteins non-covalently and covalently attached to other components in the spore wall, which is not a uniform structure. The effects of proteases, lectins, and other treatments of spores in an adhesion assay have been used to show that pre-formed glycoproteins are involved in rapid adhesion to a hydrophobic surface. There is also evidence for a rapid release of glycoproteins by spores that is also involved in adhesion and this is not an active process.

Virulence Mechanisms of Coccidioides

Coccidioides is a fungal respiratory pathogen of humans that can cause disease in both immunosuppressed and immunocompetent individuals. We describe here three mechanisms by which the pathogen survives in the hostile host environment: production of a dominant spherule outer wall glycoprotein (SOWgp) that modulates host immune response and results in compromised cell-mediated immunity to coccidioidal infection, depletion of SOWgp presentation on the surface of endospores, which prevents host recognition of the pathogen when the fungal cells are most vulnerable to phagocytic defenses, and induction of elevated production of host arginase I and coccidioidal urease, which contribute to tissue damage at sites of infection. Arginase I competes with inducible nitric oxide synthase (iNOS) in macrophages for the common substrate, L-arginine, and thereby reduces nitric oxide (NO) production and increases the synthesis of host orinithine and urea. Host-derived L-ornithine may promote pathogen growth and proliferation by providing a pool of the monoamine, which could be taken up and used for synthesis of polyamines via metabolic pathways of the parasitic cells. We have shown that high concentrations of Coccidioides- and host-derived urea at infection sites in the presence of urease produced and released by the pathogen, results in secretion of ammonia and contributes to alkalinization of the microenvironment. We propose that ammonia and enzymatically active urease released from spherules during the parasitic cycle of Coccidioides exacerbate the severity of coccidioidal infection by contributing to a compromised immune response to infection and damage of host tissue at foci of infection.

Structural analysis of linear hydroxyproline-bound O-glycans of Chlamydomonas reinhardtii—conservation of the inner core in Chlamydomonas and land plants

Linear hydroxyproline-bound O-glycans of the unicellular green alga Chlamydomonas reinhardtii were isolated from outer cell wall glycoproteins and their structure elucidated by chemical and spectroscopic methods. They consist exclusively of arabinose and galactose, the latter in the furanose form, unusual for plants. The first two arabinoses linked to hydroxyproline have the same anomeric configuration and linkage to each other as do the arabinosides isolated from land plants, suggesting that these two steps of hydroxyproline-O-glycosylation have been conserved during evolution. In a concomitant analysis of Hyp-bound O-tri- and tetraarabinans isolated from Arabidopsis thaliana, we were able to unambiguously identify the natural trans-4- l-Hyp-bound O-glycans and the artificially produced d-allo forms and to identify diagnostic signals in NMR spectra of these compounds for the first time.

Between-species analysis of short-repeat modules in cell wall and sex-related hydroxyproline-rich glycoproteins of Chlamydomonas.

Protein diversification is commonly driven by single amino acid changes at random positions followed by selection, but, in some cases, the structure of the gene itself favors the occurrence of particular kinds of mutations. Genes encoding hydroxyproline-rich glycoproteins (HRGPs) in green organisms, key protein constituents of the cell wall, carry short-repeat modules that are posited to specify proline hydroxylation and/or glycosylation events. We show here, in a comparison of two closely related Chlamydomonas species-Chlamydomonas reinhardtii (CC-621) and Chlamydomonas incerta (CC-1870/3871)-that these modules are prone to misalignment and hence to both insertion/deletion and endoduplication events, and that the dynamics of the rearrangements are constrained by purifying selection on the repeat patterns themselves, considered either as helical or as longitudinal face modules. We suggest that such dynamics may contribute to evolutionary diversification in cell wall architecture and physiology. Two of the HRGP genes analyzed (SAG1 and SAD1) encode the mating-type plus and minus sexual agglutinins, displayed only by gametes, and we document that these have undergone far more extensive divergence than two HRGP genes (GP1 and VSP3) that encode cell wall components-an example of the rapid evolution that characterizes sex-related proteins in numerous lineages. Strikingly, the central regions of the agglutinins of both mating types have diverged completely, by selective endoduplication of repeated motifs, since the two species last shared a common ancestor, suggesting that these events may have participated in the speciation process.

Induction of the gal Pathway and Cellulase Genes Involves No Transcriptional Inducer Function of the Galactokinase in Hypocrea jecorina

The Saccharomyces cerevisiae galactokinase ScGal1, a key enzyme for D-galactose metabolism, catalyzes the conversion of D-galactose to D-galactose 1-phosphate, whereas its catalytically inactive paralogue, ScGal3, activates the transcription of the GAL pathway genes. In Kluyveromyces lactis the transcriptional inducer function and the galactokinase activity are encoded by a single bifunctional KlGal1. Here, we investigated the cellular function of the single galactokinase GAL1 in the multicellular ascomycete Hypocrea jecorina (=Trichoderma reesei) in the induction of the gal genes and of the galactokinase-dependent induction of the cellulase genes by lactose (1,4-O-beta-D-galactopyranosyl-D-glucose). A comparison of the transcriptional response of a strain deleted in the gal1 gene (no putative transcriptional inducer and no galactokinase activity), a strain expressing a catalytically inactive GAL1 version (no galactokinase activity but a putative inducer function), and a strain expressing the Escherichia coli galK (no putative transcriptional inducer but galactokinase activity) showed that, in contrast to the two yeasts, both the GAL1 protein and the galactokinase activity are fully dispensable for induction of the Leloir pathway gene gal7 by D-galactose and that only the galactokinase activity is required for cellulase induction by lactose. The data document a fundamental difference in the mechanisms by which yeasts and multicellular fungi respond to the presence of D-galactose, showing that the Gal1/Gal3-Gal4-Gal80-dependent regulatory circuit does not operate in multicellular fungi.

Application of inter simple sequence repeat (ISSR) polymorphism for detection of sex-specific molecular markers in hop (Humulus lupulus L.)

Hop (Humulus lupulus L.) is dioecious species with female plants of commercial value. During breeding process it is desirable to identify the sex of hop plants at the stage of seedling. Twenty two inter simple sequence repeat (ISSR) primers were screened on female and male hop genotypes of Russian and European origin. Two ISSR primers revealed fragments specific for male plants of hop. Based on the sequences two pairs of primers were designed. These male specific sequence tagged site (STS) markers were tested on male hop accessions of Russian origin and female hop accessions of Russian, European and American origin. A high homology of male specific hop DNA sequences to expressed sequences from EMBL plants EST database was found, most of which code cell wall glycoproteins. The applicability of ISSR-PCR analysis for development of sex molecular markers in hop is discussed.

Nucleotide Sequence-Based Analysis for Determining the Molecular Epidemiology of Penicillium marneffei

The dimorphic fungus, Penicillium marneffei, is an emerging opportunistic pathogen endemic in Southeast Asia, especially for those with impaired cellular immunity such as human immunodeficiency virus-infected persons. A discriminatory and reproducible method based on the analysis of nucleotide sequences would facilitate epidemiologic investigations of this fungus. Twenty-four clinical or environmental isolates of P. marneffei obtained from China, Thailand, and Vietnam were analyzed by nucleotide sequence analysis. A total of 3,803 bp, consisting of eight nuclear gene fragments (transcription factor [AbaA], catalase [CpeA]], homodomain transcription factor [StlA], isocitrate lyase [Icl1], polyaromatic amino acid biosynthesis [PAA], NADH-dependent glutamate synthase [NGS], lovastatin nonaketide synthase [LNS], a cell wall mannoprotein [MP1], and a gene fragment of the cytochrome oxidase subunit 1 gene [COX1] of the P. marneffei mitochondrial genome) were amplified by PCR and then sequenced. No polymorphic sites within the Cox1 gene fragment were observed. Likewise, no nucleotide sequence polymorphisms were observed for three gene fragments: StlA, AbaA, and NGS. Seven single-nucleotide polymorphisms were observed for three gene fragments, Icl1, CpeA, and PAA, providing only a low degree of discriminatory power (D = 0.747). In contrast, the gene fragment for an antigenic cell wall glycoprotein, MP1, a useful immunologic marker for infection, was observed to be highly polymorphic with 12 different MP1 types (D = 0.887). Single-nucleotide polymorphisms were observed at 21 different locations in the MP1 gene fragment. Indels of 3, 21, 24, and 42 bp were observed and were in frame for protein translation. The relatively high degree of MP1 polymorphisms suggests the sequence is rapidly evolving in order to evade host immune responses. After all polymorphic gene sequences were combined, a high degree of genetic variation was observed (D = 0.949) for a total of 16 different haploid sequence types with 11 genotypes represented by single isolates. Phylogenetic analysis detected clusters composed of isolates obtained only from China or Thailand, as well as clusters with a combination of isolates from these two countries, indicating some mixing or common descent. Identical sequences were observed for isolates passed in vitro for 8 weeks, suggesting good reproducibility. The low degree of nucleotide diversity in housekeeping and regulatory genes suggests the recent emergence and spread as a species or an evolutionary bottleneck. In summary, multilocus sequence typing demonstrated a high degree of discriminatory power and reproducibility and may provide a robust and reliable adjunct method for genotyping isolates of P. marneffei and facilitating interlaboratory comparisons.

The fission yeast Map4 protein is a novel adhesin required for mating

Cell adhesion is required for many cellular processes. In fungi, cell–cell contact during mating, flocculation or virulence is mediated by adhesins, which typically are glycosyl phosphatidyl inositol (GPI)-modified cell wall glycoproteins. Proteins with internal repeats (PIR) are surface proteins involved in the response to stress. In Schizosaccharomyces pombe no adhesins or PIR proteins have been described. Here we study the S. pombe Map4p, which defines a new class of surface protein that is not GPI-modified and has a serine/threonine rich domain and internal repeats that differ from those present in PIR proteins. Map4p is a mating type-specific adhesin required for mating in h + cells and enhances cell adhesion when overexpressed.

Saccharomyces cerevisiae adaptation to weak acids involves the transcription factor Haa1p and Haa1p-regulated genes

The understanding of the molecular mechanisms that may contribute to counteract the deleterious effects of organic acids as fungistatic agents is essential to guide suitable preservation strategies. In this work, we show that the recently identified transcription factor Haa1p is required for a more rapid adaptation of a yeast cell population to several weak acid food preservatives. Maximal protection is exerted against the short-chain length acetic or propionic acids. The transcription of nine Haa1p-target genes, many of which are predicted to encode membrane proteins of unknown or poorly characterized function, is activated under weak acid stress. The Haa1-regulated genes required for a more rapid yeast adaptation to weak acids include TPO2 and TPO3, encoding two predicted plasma membrane multidrug transporters of the major facilitator superfamily, and YGP1, encoding a poorly characterized cell wall glycoprotein. The acetic acid-induced prolongation of the lag phase of unadapted cell populations lacking HAA1 or TPO3, compared with wild-type population, was correlated with the level of the acid accumulated into the stressed cells.

Monoclonal Immunoglobulin G1 Directed againstAspergillus fumigatusCell Wall Glycoprotein Protects against Experimental Murine Aspergillosis

Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, which, being the outermost part of the cell, mediates the host-fungus interplay. For these reasons much effort has focused on the discovery of useful inhibitors of cell wall glucan, chitin, and mannoprotein biosynthesis. In the absence of a wide-spectrum, safe, and potent antifungal agent, a new strategy for antifungal therapy is directed towards the development of monoclonal antibodies (MAbs). In the present study the MAb A9 (immunoglobulin G1 [IgG1]) was identified from hybridomas raised in BALB/c mice immunized with cell wall antigen of Aspergillus fumigatus. The immunoreactive epitopes for this IgG1 MAb appeared to be associated with a peptide moiety, and indirect immunofluorescence microscopy revealed its binding to the cell wall surface of hyphae as well as with swollen conidia. MAb A9 inhibited hyphal development as observed by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (25.76%), reduced the duration of spore germination, and exerted an in vitro cidal effect against Aspergillus fumigatus. The in vivo protective efficacy of MAb A9 was also evaluated in a murine model of invasive aspergillosis, where a reduction in CFU (>4 log(10) units) was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2 x 10(5) CFU/ml) and where enhanced mean survival times (19.5 days) compared to the control (7.1 days) and an irrelevant MAb (6.1 days) were also observed.

FLO11-Based Model for Air-Liquid Interfacial Biofilm Formation bySaccharomyces cerevisiae

Sardinian wine strains of Saccharomyces cerevisiae used to make sherry-like wines form a biofilm at the air-liquid interface at the end of ethanolic fermentation, when grape sugar is depleted and further growth becomes dependent on access to oxygen. Here, we show that FLO11, which encodes a hydrophobic cell wall glycoprotein, is required for the air-liquid interfacial biofilm and that biofilm cells have a buoyant density greater than the suspending medium. We propose a model for biofilm formation based on an increase in cell surface hydrophobicity occurring at the diauxic shift. This increase leads to formation of multicellular aggregates that effectively entrap carbon dioxide, providing buoyancy. A visible biofilm appears when a sufficient number of hydrophobic cell aggregates are carried to and grow on the liquid surface.